Characterization of feline nonsteroidal anti-inflammatory drug activated gene-1 (fNAG-1) and its protective function in kidney cells

Abstract

Background: Domestic cats are susceptible to obesity and chronic renal failure, leading to significant health risks. Nonsteroidal anti-inflammatory drug-activated gene (NAG-1), also known as growth differentiation factor 15 (GDF15), is a member of the transforming growth factor-β superfamily and has been associated with anti-obesity properties and preservation of kidney function. While the NAG-1 sequence has been extensively studied in several species, a comprehensive understanding of feline NAG-1 remains limited. This study aimed to investigate the nucleotide sequence of feline NAG-1 and its biological role in kidney protection through in-vitro experiments.

Methods: The feline NAG-1 cDNA was isolated from the feline uterus, and its sequence was analyzed and compared to sequences from other species, including humans. Expression patterns of feline NAG-1 in various tissues, particularly the liver and kidney, were determined. Furthermore, the effects of different phytochemicals and NSAIDs known to induce NAG-1 expression were assessed using Crandell-Rees Feline Kidney (CRFK) cells.

Results: The analysis revealed that feline NAG-1 shares similarities with human NAG-1 and exhibits high expression levels in the liver and kidney of cats. Treatment with tolfenamic acid, quercetin, and resveratrol significantly increased NAG-1 expression in CRFK cells. Subsequently, CRFK cells overexpressing feline NAG-1 were utilized to investigate the functional roles of NAG-1 in feline kidney health. High-content screening analysis demonstrated that NAG-1 overexpression in cat kidney cells enhanced mitochondrial membrane potential, reduced reactive oxygen species (ROS) generation in both whole cells and mitochondria, and downregulated the expression of Bax, a pro-apoptotic protein, under conditions of ROS-induced stress. These findings indicate the renoprotective role of NAG-1.

Conclusion: This study highlights the significant role of NAG-1 in feline kidney cells, revealing its high expression in the liver and kidney and demonstrating its protective effects on kidney function. These results underscore the potential of NAG-1 as a key factor in kidney protection. Future research should focus on further elucidating the molecular pathways involved and exploring therapeutic strategies to harness NAG-1 for managing obesity-related renal dysfunction in cats.

Keywords: Cat; Kidney; Mitochondria; NAG-1; Obesity; Phytochemicals.

Fig. 1

The nucleotide sequence (shown on top) and the deduced amino acid sequence (shown at the bottom) of feline NAG-1 were examined. In the sequences displayed, the portions highlighted in red indicate variations when compared to the reference sequence from the NCBI GenBank (XM_023247515.1)

Fig. 2

Comparison of NAG-1 protein sequence for various species. (A) Multiple alignments of NAG-1 amino acid sequence by species. The positions that are 100% conserved in the alignment are marked with an asterisk (*). A bold letter with a gray background indicates RXXR motifs. (B) Phylogenetic tree based on NAG-1 amino acid sequence homology. Except for Felis catus, all protein sequence information was obtained by NCBI GenBank (Canis lupus, XP_038284083.1; Sus scrofa, NP_001167527.1; Homo sapiens, NP_004855.2; Pan troglodytes, XP_009433302.1; Rattus norvegicus, NP_062089.1; Mus musculus, NP_001317616.1). The scale bar indicates the number of substitutions per site

Fig. 3

NAG-1 expression in cat tissues. (A) Feline NAG-1 expression after transient transfection of feline NAG-1 cDNA into CRFK cell lines. Feline NAG-1 and GAPDH expression were detected by RT-PCR. The empty vector (e.v) indicates transfection of intact pCDNA3.1 plasmid without any insert cDNA. Results were expressed as mean ± SD (n = 3). The statistical analysis was analyzed using student t’s test, **P < 0.01, ***P < 0.001 compared with negative control. (B) Expression profiles of NAG-1 in feline tissues including liver, kidney, spleen, and skin

Fig. 4

Effects of phytochemicals and NSAIDs in feline kidney cell line. Each compound diluted in serum-free media was treated with CRFK cells and incubated for 24 h. All compounds were used at 50 µM concentration. Feline NAG-1 and GAPDH expression were performed by (A) RT-PCR and (B) qRT-PCR. Results were expressed as mean ± SD (n = 3). The statistical analysis was calculated using One-way ANOVA with Dunnett’s comparison test, *P < 0.05, **P < 0.01, ***P < 0.001 compared with negative control

Fig. 5

The reno-protection effect of NAG-1 overexpression in cat kidney epithelial cell line. (A) The relative fluorescence intensity of mitochondrial membrane potential in empty vector (e.v.) transfected- and feline NAG-1 transfected cell line. To assess transfection efficiency, all cells, regardless of whether they were transfected with the empty vector (e.v.) or the fNAG-1 expression vector, were additionally co-transfected with a GFP (green fluorescent protein) expression vector. Results are expressed as mean ± SD. The statistical analysis was calculated using student’s t-test method, ***P < 0.001 compared with e.v. transfected cells. (B) The relative intensity of H2DCFDA/Hoechst 33,342, and the representative images of the ROS accumulation in MMI induced ROS cells. The cellular ROS scavenging activity was observed in cells transiently transfected with NAG-1 cDNA following exposure to 4 mM MMI for 48 h to induce oxidative stress. While NAG-1 overexpression did not significantly alter basal ROS levels, it demonstrated a protective effect against ROS-induced damage in kidney cells. Images were acquired, and quantitative data were analyzed using high-content screening (HCS) at 200x magnification. The statistical analysis was measured using One-way ANOVA with Tukey’s comparison test (*P < 0.05, ***P < 0.001 compared with e.v transfected cell; ^#P < 0.05 compared with fNAG-1 transfected cells with 4 µM MMI).

Fig. 6

The potential of NAG-1 to reduce the mitochondria ROS generation in CRFK cells. (A) The mitochondria ROS assay was performed using mitoSOX, mitochondria superoxide indicator. After transfection CRFK cells with empty vector (e.v.) and feline NAG-1 plasmid, living cells were stained with mitoSOX for 10 min. Red: MitoSOX. The pictures were captured, and quantitative results were analyzed using high content screening (HCS) with 400x magnification. Results are expressed as mean ± SD. The statistical analysis was calculated using One-way ANOVA with Tukey’s comparison test, ***P < 0.001, ****P < 0.0001 compared with e.v transfected cell; ^##P < 0.01, ^####P < 0.0001 compared with fNAG-1 transfected cells with 4 µM MMI). (B) Attenuation of Bax protein expression in fNAG-1 overexpression cell lines, compared with control, after treating with ROS-induced compound. There was no differential expression of Bax protein in both group in normal condition, whereas NAG-1 transfected cells show lower Bax expression after inducing ROS by 4 µM MMI, compared with their control. Results are expressed as mean ± SD. The statistical analysis was performed using ANOVA with Tukey’s comparison test, ***P < 0.001, compared with e.v transfected cell; ^###P < 0.001 compared with fNAG-1 transfected cells with 4 µM MMI